Femtogram-level detection of Clostridium botulinum neurotoxin type A by sandwich immunoassay using nanoporous substrate and ultra-bright fluorescent suprananoparticles.
Femtogram-level detection of Clostridium botulinum neurotoxin type A by sandwich immunoassay using nanoporous substrate and ultra-bright fluorescent suprananoparticles.
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使用纳米多孔基质和超亮荧光超纳米颗粒，通过夹心免疫测定法对 A 型肉毒杆菌神经毒素进行飞克级检测。
DOI：
10.1016/j.bios.2012.08.063
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发表时间：
2013
影响因子：
12.6
通讯作者：
S. Gangopadhyay
中科院分区：
文献类型：
--
作者：
S. Bok;V. Korampally;Charles M. Darr;W. Folk;L. Polo;K. Gangopadhyay;S. Gangopadhyay
我们报告了一种简单，强大的荧光生物传感器的超灵敏检测肉毒梭菌神经毒素A型（BoNT/A）在复杂的，现实世界的媒体。通过组合使用超亮、光稳定的染料掺杂纳米颗粒（DOSNP）标签和高表面积纳米多孔有机硅酸盐（NPO）薄膜来实现高固有信号放大。合成了直径为22 nm的DOSNP，其游离染料荧光超过200倍当量，并以每个抗体90个染料的平均取代度与抗体缀合，与常规染料标记的抗体相比，代表了一个数量级的增加。NPO膜被设计成在荧光团所在的表面处形成相长干涉。此外，DOSNP标记的抗体与NPO膜增加了表面粗糙度，导致漫散射，导致散射强度比染料标记的抗体与NPO膜高24%。这些底物用于固定针对BoNT/A的捕获抗体，其通过DOSNP标记的信号抗体进一步定量。光学效应的组合增强了荧光，因此显著增强了信噪比。在PBS缓冲液中检测到BoNT/A，在4小时内降至21.3fgmL− 1。然后将该检测方法扩展到几种复杂介质，发现4小时的检测限分别为橙子汁中的145.8fgmL− 1和自来水中的164.2fgmL− 1，与其他酶联免疫吸附测定（ELISA）的检测限相比至少提高了两个数量级。因此，该测定证明了一种快速、超低水平检测BoNT/A和其他分析物的新方法。
We report a simple, robust fluorescence biosensor for the ultra-sensitive detection of Clostridium botulinum Neurotoxin Type A (BoNT/A) in complex, real-world media. High intrinsic signal amplification was achieved through the combined use of ultra-bright, photostable dye-doped nanoparticle (DOSNP) tags and high surface area nanoporous organosilicate (NPO) thin films. DOSNP with 22nm diameter were synthesized with more than 200 times equivalent free dye fluorescence and conjugated to antibodies with average degree of substitution of 90 dyes per antibody, representing an order of magnitude increase compared with conventional dye-labeled antibodies. The NPO films were engineered to form constructive interference at the surface where fluorophores were located. In addition, DOSNP-labeled antibodies with NPO films increased surface roughness causing diffuse scattering resulting in 24% more scattering intensity than dye-labeled antibody with NPO films. These substrates were used for immobilization of capture antibodies against BoNT/A, which was further quantified by DOSNP-labeled signal antibodies. The combination of optical effects enhanced the fluorescence and, therefore, the signal-to-noise ratio significantly. BoNT/A was detected in PBS buffer down to 21.3fgmL−1in 4h. The assay was then extended to several complex media and the four-hour detection limit was found to be 145.8fgmL−1in orange juice and 164.2fgmL−1in tap water, respectively, demonstrating at least two orders of magnitude improvement comparing to the reported detection limit of other enzyme-linked immunosorbent assays (ELISA). This assay, therefore, demonstrates a novel method for rapid, ultra-low level detection of not only BoNT/A, but other analytes as well.
影响因子：
2.9
作者：
STAROS, JV;WRIGHT, RW;SWINGLE, DM
通讯作者：
SWINGLE, DM
影响因子：
8.4
作者：
Memisevic, Jasenka;Korampally, Venumadhav;Gangopadhyay, Shubhra;Grant, Sheila A.
通讯作者：
Grant, Sheila A.